Changes in β3 receptor levels in diabetes mellitus and infravesical obstruction-induced cystopathy in rat bladders

Authors

Abstract

Aim This study aims to immunohistochemically determine potential changes in the number of β3 receptors caused by diabetes and bladder outlet obstruction (BOO), an ailment whose frequency increases with age.
Materials and Methods Four distinct groups, each comprising 12 rats, were established: a control group, a diabetic group, an obstructive group, and a diabetic obstructive group. To induce moderate obstruction, a 5 Fr catheter was utilized. For moderate diabetes induction, a dose of 50 mg/kg STZ was intraperitoneally administered to the rats. Over 8 weeks, the rats’ blood sugar and body weight levels were closely monitored. After euthanasia, the rats’ bladder tissues were extracted for subsequent histological and immunohistochemical assessments. Statistical analyses were conducted to compare the results, with significance attributed to p-values < 0.05.
Results The histopathological examination revealed notable variances among the groups concerning urothelial hyperplasia and inflammation. Moreover, a discernible increase in intensity of β3 receptor staining was noted in the obstructive group when compared to the control group, whereas a decrease was observed in both the diabetic and diabetic obstructive groups.
Discussion Alterations in receptor count may not necessarily correspond to equivalent changes in receptor functionality. Consequently, a comprehensive evaluation of receptor function necessitates diverse in vivo and in vitro investigations.

Keywords

Introduction

The sympathetic nervous system regulates urine storage in the bladder. Beta receptors are more dominant in the detrusor muscle than alpha receptors, and the primary function of β- adrenoceptors in the bladder is to provide smooth muscle relaxation and an increase in bladder compliance during the micturition cycle’s filling phase [1]. It has been reported that mRNA expression encoding β3 adrenergic receptors constituted 97% of total mRNA expression encoding β3 adrenergic receptors and that β3 adrenergic agonists achieve the most effective relaxation among these receptors [2].
The changes in abnormal bladder activity such as increased spontaneous activity [3], hypersensitivity to cholinergic agonists [4], detrusor denervation [5] , secondary excitatory neurotransmitters such as ATP [6] , and increased electrical excitability of the muscle [7], are difficult to classify. Diabetes also leads to peripheral autonomic neuropathy and detrusor overactivity is a known pathology.
This study aimed to immunohistochemically detect any possible changes in the level of β3AR in the bladder in the pathological process caused by diabetes and obstruction. According to our knowledge, we share the first information about these two pathological processes, which are frequently seen with age and sometimes occur together, with the medical literature.

Materials and Methods

Experimental Animals
Wistar-albino male rats weighing 300-350 g obtained from Canakkale Onsekiz Mart University (COMU) Experimental Medicine Research and Application Center were used and divided into four groups: 1- Control, 2- Diabetic, 3- Partial obstructive, 4- Diabetic-partial obstructive [n=12 for each group]. The animals had free access to food and water and kept in separate cages throughout the experiment. During the study, the rats were housed in the COMU Experimental Animals Unit, each in a single cage, where the temperature (22±1ºC) and lighting [light between 07.00-19.00 hours and dark between 19.00-07.00 hours] were kept under control.
Experimental Protocol
Creating Partial Obstruction
After anesthesia, the abdomen was opened through a suprapubic median incision to create a moderate partial obstruction. A 5F ureteral catheter was placed in the bladder neck, and the bladder neck was ligated with a non-absorbable 3/0 silk suture, protecting the surrounding vessels. Eight weeks after partial obstruction, the rats were sacrificed with high-dose anesthetic. Cystectomy specimens were sent for immunohistochemical analysis.
Creation and Follow-Up of Diabetes
50 mg/kg streptozotocin (STZ) was administered intraperitoneally to 24 rats designated to develop diabetes, and blood glucose was measured 4 days later by taking blood samples from the tail vein. The study excluded rats with blood glucose levels less than 200 mg/dl. Partial obstruction was applied to half of the rats with diabetes and they were monitored for 8 weeks.
Histopathological Methods and Immunohistochemical Staining For all groups, Hematoxylin and Eosin (H&E) staining and Immunohistochemical staining with Rabbit β3AR antibodies were applied to sections obtained from prepared paraffin blocks. The stained sections were evaluated under a light microscope.
While urothelial hyperplasia was evaluated by the histologist, if the urothelium had fewer than 2-3 layers, it was interpreted as no hyperplasia, 3-5 layers as weak, and more than 5 layers as severe hyperplasia. When assessing inflammation, the number of inflammatory cells was taken into account and classified as absent, low, moderate and high. Absence of staining when assessing β3AR epithelial staining Score 0, localized small areas Score 1, more extensive areas (staining mostly in umbrella cells) Score 2 and more extensive areas (staining in all layers of the epithelium) Score It was rated as Score 3. β3AR staining in the connective tissue between the suburothelium and the detrusor was evaluated as Score 1 for low staining and Score 2 for increased staining.
The same histologist performed the histopathological examination without knowing which tissue samples belonged to which group, and by random selection from tissue samples (blind evaluation).
The photos were taken using the Zeiss Scope A1 Light microscope with ZEN blue edition software and Axiocam 503 color camera attachment.
Statistical Analysis
Statistical analyses and graphical outputs were generated using the JAMOVI software. One of the variables in this study is the group in which the rats are included, and the other variable is some parameter measurements obtained from the rats. The Chi-square test was used in the analysis of categorical variables belonging to control, diabetic, obstructive and diabetic-obstructive groups. Since the number of subjects in the groups was 5 or less, Fisher’s exact test was used. A value of p<0.05 was accepted as the level of significance.
The Kruskal-Wallis Test was used to compare bladder weights, body weights and blood sugars based on the experimental groups of rats.
Ethical Approval
This study was approved by the COMU Animal Experiments Local Ethics Committee (Date: 2021-10-22, No: 2021/09).

Results

At the end of the experiment, 8 rats from the control, diabetic and obstructive groups, and 7 rats from the diabetic-obstructive group were evaluated.
The rats’ blood sugar levels, body weights and bladder weights Blood glucose values in rats in all groups statistically differed from each other (p:0.000). At the beginning of the experiment, there were no statistically significant difference in pre- experiment body weights of rats in all groups (p:0.054). At the end of the experiment, it was found that the body weights of the rats in the control, diabetic, obstructive and diabetic- obstructive groups statistically significantly differed (p:0.003). It was found that the bladder weights of the groups statistically significantly differed (p:0.000) (Table 1).
Table 1. Blood Sugar (BS), distribution of rat weight and bladder weights at the beginning and end of the experiment (p<0.05 was considered significant, the results are given as mean±standard deviation)
Histopathological Data of Rats
A significant difference was observed for the presence of epithelial hyperplasia (p=0.000). Hyperplasia was not observed in rats in the control group, but it was observed in all rats in the diabetic obstructive group. When the groups were compared in terms of mild and severe hyperplasia, it was found that there was a significant difference between the diabetic obstructive group and the other groups (Figure 1).
It was found that there was a statistical difference for the presence of inflammation (p=0.000). Inflammation was observed in all rats in the diabetic obstructive group. In the case of moderate inflammation, between the diabetic- obstructive group and each of the diabetic and obstructive groups. (Inflammation was evaluated at 4 levels as absent, low, moderate and high, and this sentence was commented in terms of moderate inflammation-for informational purposes- we prefer to use our expression) In the case of very severe inflammation, it was found that there was a significant difference between the control group and the diabetic and obstructive groups.
β3AR Immunohistochemical Staining Data
The presence of β3AR immunostaining in the connective tissue between the urothelium, detrusor and suburothelium and detrusor layer was investigated and staining was monitored. No significant difference was observed between the groups in terms of β3 receptor staining in the bladder urothelium (p=0.553) (Figure 2a). However, although it was not statistically significant, the degree of β3 receptor staining in the bladder urothelium of obstructed rats was observed to be higher than in the other groups. Diffuse staining in facet cells was more apparent in the obstruction and diabetes groups (Figure 3).
A significant difference in β3 receptor staining was observed between the groups when the connective tissue between the suburothelium and detrusor was evaluated [p=0.004]. Intensive staining was observed in all rats in the control group, while a decrease in receptor staining was observed in the connective tissue between the suburothelium and the detrusor of the rats in the diabetic group and the diabetic obstruction group. No statistically significant difference was observed between the groups (Figure 2b). When compared with the control group, a decrease in staining intensity was observed in the obstructive group, the diabetic group, and the diabetic obstructive group, respectively (Figure 3).

Discussion

We intended to investigate DM and BPH’s effect on the level of β3 receptor in the bladder when they were present separately and together in our study. We attempted to create a mild diabetes model using 50 mg/kg STZ, as Magadmi et al. did in their study [8]. Since studies using diabetic models revealed that bladder dysfunction develops over time, evaluations were performed in the 8th week, when the chronic diabetes changes would be considered apparent [9].
The most commonly used methodological approach to create the animal model of BOO is partial urethral obstruction. Although animal models do not fully replicate clinical symptoms, studies have shown that rats with partial urethral obstruction show urinary emptying and storage symptoms similar to bladder outlet obstruction in humans [10].
According to Lin et al., BOO causes an increase in inflammation, oxidative stress, ischemia and hypoxia in the bladder, resulting in impaired bladder function [11]. As a result of partial obstruction of the bladder, a significant increase in bladder weight is observed in the early period as a result of inflammation due to various growth factors, uroepithelial hyperplasia, smooth muscle hypertrophy, type 3 collagen synthesis and storage. Although these morphological changes are expected in both pathologies, the results should also be related to the duration. In our study, the changes in the 8-week period were evaluated. Several studies have reported increased urine output as a result of diabetes-related diuresis and bladder weight increases as a result of developing detrusor hypertrophy [9, 12]. In our study, the blood glucose levels of the control and obstructive groups were found to be within the normal range, and the bladder and body weights of the rats in these groups were increased. When we compared all groups, we found that the bladder weight increased statistically significantly in all groups. It was thought that bladder weight increased as a result of the physiopathological processes caused by diabetes and obstruction.
Limberg et al. have suggested that β3ARs were located in the urothelium instead of the detrusor. They discovered pale staining for β3AR throughout the entire bladder wall, including detrusor muscle cells, urothelium, myofibroblasts, and nerve- like structures of unknown origin [13]. Coelho et al. discovered β3AR-IR [immunoreaction] in the mucosa and muscle layer of the bladder along the trigone, lateral wall and dome. The majority of β3AR-IRs were discovered in varicose linear structures, the density of which is much higher in the suburothelium than in the muscle layer. Linear structures were observed primarily in the suburothelium and deep suburothelial regions, and they did not appear to penetrate the urothelial layer. In the muscle layer, β3AR-IR was found on the varicose linear structures running along the surface of the smooth muscle bundles. The structures’ density was reported to be lower than that of the suburothelium and there was no immunoreaction in smooth muscle cells. The detection of β3AR-IR in cholinergic nerve fibers running along the suburothelium and detrusor layer, as well as the absence of β3AR-IR in smooth muscle cells, is the most important finding of the study of Coelho et al. [14]. Studies demonstrating the efficacy of β3 receptor agonists in BOO-induced hyperactive bladder are also added to the literature [15]. In our study, consistent with the literature, β3 receptor staining was observed in the urothelium, suburothelial connective tissue and muscle tissue in the control group. Consistent with the literature, no staining was found in the detrusor muscle cells. We found no studies on diabetes and β3 receptor levels in the obstructive model in the literature. In addition, we found no statistically significant differences in β3 receptor staining in the urothelium in our diabetes, obstructive, and diabetic obstructive models. However, β3 staining was more prevalent in the obstructive group than in the other groups. In the staining of the suburothelial connective tissue and the muscle tissue, a significant staining difference was observed between the control group and the diabetic and diabetic obstructive groups. However, no significant difference was observed when comparing the control with obstructive group and the diabetic group with the diabetic-obstructive group. These findings demonstrate that β receptor changes can differ in various pathologies.
In vitro urine strip experiments have shown that β3AR agonists relax smooth muscle regardless of the presence of other cell types in the urothelium and lamina propria [16]. Thus, the effect of β3AR agonists on the micturition reflex may involve receptors located not only in smooth muscle but also in the sensory system, including the urothelium. Similarly, our findings suggest that β3AR agonists affect urothelium and suburothelium cells, as well as muscle cells. Although the majority of the effects of β3AR agonists are attributed to the activation of smooth muscle receptors, recent studies using RT-PCR and/or immunohistochemistry have detected all βAR subtypes in the urothelium. In our study, we evaluated the receptors using the immunohistochemical method and observed various degrees of staining in the groups. By RT-PCR, mRNA expressions at levels comparable to or exceeding those expressed in smooth muscle have been reported for all βARs in the urothelium [17]. The results of our immunohistochemical study appear to be consistent with the PCR result.
The efficacy of relaxation factors in the urothelium has been demonstrated in a previous study in rat bladders by forming groups in which the urothelium either intact or removed. [18]. A recent study conducted on the porcine bladder by Masunaga et al. revealed that β3 receptors in the urothelium contribute to the inhibitory effect of β receptors in smooth muscle via the urothelium. In our study, we discovered evidence that the urothelium may have an inhibitory effect by relaxing the detrusor.
When we evaluated all groups, the number of β3 receptors in the urothelium was higher in other groups than in the control group, and a decrease was observed in the suburothelium in other groups compared to the control group. The total number of β3 receptors increased in the obstructive group, that is, in the BOO model, compared to the control group, and the increase in the number of receptors suggested an increase in responsiveness to the β3 agonist. The decrease in the number of receptors in the diabetic and diabetic obstructive groups compared to the control group suggested a decrease in responsiveness to the β3 agonist. The decrease in the number of receptors does not provide a suggestion about the increase or decrease in the receptor function. For more information, electrophysiological studies [organ baths, etc.] are needed.

Limitations

Experimental approaches, especially animal studies, cannot fully model the physiopathological processes in humans and should be considered preliminary studies. The changes revealed in the coexistence of these two important pathologies should be supported by in-vivo experiments and studies evaluating their effects on receptor sensitivity. It is considered that controlled clinical studies for the pathologies that are the subject of this study are needed.

Conclusion

Similar to the results we have obtained in the literature, we found β3 receptor staining in all layers of the bladder and no staining in detrusor smooth muscle cells. Our findings suggest that β3 receptors in the urothelium and suburothelial layer play an active role in detrusor smooth muscle relaxation.
Our study showed that the differences in the ratio of β3 receptors in the urothelial and suburothelial connective tissues may be factors that have gained different importance in various pathologies and remain unexplained. Histopathological changes were also observed to play a role. Especially the significant decrease in β3 receptor staining in the connective tissue between the suburothelium and detrusor of the diabetic and diabetic-obstructive groups compared to the control group between the suburothelium and the detrusor, suggested that it may play an important role in cystopathy, which may increase over time in the diabetic process, and may contribute to shaping the medical approaches involving β receptors in the symptomatic treatment of BOO.

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